Cell-free synthesis of deacetoxycephalosporin C

Abstract

A cell-free process for converting isopenicillin N, delta -(L- alpha -aminoadipyl)-L-cysteinyl-D-valine (hereinafter "LLD") and 6-substituted derivatives thereof to deacetoxycephalosporin C (DACPC) by the following reaction sequence: <IMAGE> is disclosed. In addition to the starting material, the reaction system includes ATP and a fresh extract of Cephalosporium acremonium prepared and used in a manner to preserve the racemase agent or agents necessary for conversion of the isopenicillin N to penicillin N, a necessary intermediate step in the process.

We claim: 
     
       1. A process for producing deacetoxycephalosporin C or 6-substituted derivatives thereof comprising the steps of: A. providing a starting material selected from the group consisting of: (a) a derivative of L-α-aminoadipyl-L cysteinyl-D-valine of the formula: ##STR8##  and (b) an isopenicillin derivative of the formula: ##STR9##  where R is selected from the group consisting of hydrogen, methyl, ethyl, propyl, and isopropyl;     B. preparing a cell-free extract of Cephalosporium acremonium, the preparation of the extract being such as to preserve the operability of an enzyme contained therein capable of inverting the aminoadipyl side chain of the penicillin molecule from the L to the D conformation;   C. contacting the extract and the starting material in a reaction zone while promoting oxygen transfer;   D. providing ATP as an energy source to said reaction zone; and   E. allowing a component of the extract to react with said starting material for a sufficient amount of time to produce a cephalosporin compound of the formula: ##STR10##   
     
     
       2. The process as set forth in claim 1 wherein R is hydrogen. 
     
     
       3. The process as set forth in claim 1 wherein the extract used in step C has not been homogenized or frozen. 
     
     
       4. The process as set forth in claim 1 wherein the ATP utilized is regenerated by a salt-free ATP regenerating system comprising a phosphate donor and a phosphotransferease enzyme. 
     
     
       5. The process as set forth in claim 4 wherein the phosphate donor is phosphoenol pyruvate and the phosphotransferase enzyme is pyruvate kinase. 
     
     
       6. The process as set forth in claim 1 wherein said cell-free extract is a fresh extract prepared by treating a Cephalosporium acremonium culture with a lysing enzyme. 
     
     
       7. The process as set forth in claim 6 wherein said cell-free extract is made by treating Cephalosporium acremonium cells with endo β(1→3) glucanase, endo β(1→4) glucanase, and zymolyase. 
     
     
       8. The process as set forth in claim 1 wherein oxygen transfer is promoted by shaking the reaction components in the reaction zone. 
     
     
       9. The process as set forth in claim 1 wherein sucrose and trace concentrations of KCl and MgSO 4  are included in the reaction system, and the system is buffered to about pH 7.2.